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ALTERED INTRACELLULAR TRAFFICKING OF NR1 SUBUNIT IN SCHIZOPHRENIA
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Abstracts of the 3rd Biennial Schizophrenia International Research Conference / Schizophrenia Research 136, Supplement 1 (2012) S1–S375
to many cellular processes including the recycling of NMDA receptors and through this mechanism may contribute to the NMDA receptor hypofunction described in schizophrenia. It is responsible for the uptake of cargos as part of a clathin coated vesicles which, once endocytosed and uncoated form early endosomes. These can either recycle directly back to the plasma membrane, transfer to recycling endosomes, or enter the late endosomal/lysomal pathway for degradation. Apart from their role in CME, clathrin-interacting proteins are involved in a wide network of cellular processes, including endosomal sorting, lysosome biogenesis, antigen presentation, and cell migration. While trafficking changes in schizophrenia have been considered previously, a specific role for the clathrin interactome has not been considered. We integrate recent evidence implicating CME and related cellular trafficking mechanisms in the pathophysiology of psychotic disorders. Specifically, we review the proteomic and genomic literature, including our own data, for a potential role of CME, and membrane trafficking generally, in psychosis and we searched for evidence, linking CME with key features of psychosis pathophysiology, namely synaptic function, white matter integrity, and neurodevelopment, and studies linking the pharmacology of antipsychotic drugs with endocytic mechanisms. Proteomic and genomic findings implicate proteins and genes of the clathrin interactome. Additionally, several important candidate genes for schizophrenia, such as dysbindin, are involved in processes closely linked to CME and membrane trafficking. Furthermore, many antipsychotic drugs have been shown to affect clathrin-interacting proteins. Based on unbiased systems proteomics and genetic studies, we propose that there is now solid evidence for disturbances of proteins interacting with the membrane vesicle coat protein clathrin in psychosis (1). We propose that the targeted pharmacological manipulation of the clathrin interactome may offer fruitful opportunities for novel treatments of schizophrenia.
ALTERED INTRACELLULAR TRAFFICKING OF NR1 SUBUNIT IN SCHIZOPHRENIA Chang-Gyu Hahn, Anamika Banerjee, Matthew L. MacDonald, Karin E. Borgmann-Winter Department of Psychiatry and Pharmacology, University of Pennsylvania, Philadelphia, PA, USA Multiple lines of evidence support the premise of attenuated NMDA receptor (NR) function in schizophrenia (SCZ), yet the molecular underpinnings for such receptor dysfunction are largely unknown. Synaptic NR complexes reside in the postsynaptic density (PSD) and consist of NR subunits, scaffolding proteins and many other signaling proteins. Thus, the overall receptor function will be determined by the abundance of NR complexes in the PSD, their protein composition and post-receptor signaling activity. Despite multiple confounds, postmortem brains of patients may harbor disease specific characteristics. Evaluation of receptor activity in postmortem brains, however, has been a technical challenge. In this study, we evaluated NR complexes in the postmortem prefrontal cortex (PFC) of 14 pairs of SCZ subjects and their matched pairs with respect to the abundance and synaptic targeting of NR subunits. MK-801 is a non-competitive open channel blocker whose binding to NRs is relatively irreversible. Thus, MK-801 binding in synaptic membranes could reflect the abundance of membrane bound NR complexes. We found that Bmax for MK-801 was enhanced by about 30% (p≤0.017), while the dissociation constant, KD, was also increased in the SCZ group (p≤0.01). This increase in MK-801 binding could be accompanied by increases in NRs and NR complex proteins in the PSD. To address this, we obtained subcellular fractions enriched for the PSD (called PSD fractions henceforth). We found that NR1 subunit was significantly enhanced in the SCZ group compared to the control (p values ≤0.03), while NR2A and NR2B subunits and scaffolding proteins were unaltered. The increase in NR1 in the PSD could result from an up-regulation of the protein or from altered intracellular trafficking. We quantified NR1 levels in the whole tissue homogenate, synaptic membrane, vesicular and parasynaptic fractions. We found no between group differences of NR1 expression in all fractions except a trend for a decrease in para-synaptic fractions. Intracellular trafficking and subcellular localization of NR1 is tightly regulated by the expression of NR1 isoforms, C1, C2 and C2’. The C2’ isoform has higher affinity for PSD-95 and the NR1: PSD-95 association facilitates the insertion of the complex into the PSD. To test the role of C2’, PSD fractions derived from the PFC of patients and their matched controls
were immunoprecipitated for Pan’-NR1 and immunoblotted for NR1-C2’. We observed an increase in the C2’ splice variant of NR1 in schizophrenic samples with respect to pan NR1 (p≤0.04), suggesting that increased NR1 in the PSD and resultant increase in NR1: PSD-95 associations could be from an over-expression of C2’. Together, these results demonstrate that there are more NR complexes in synaptic membranes in SCZ, which is accompanied by altered intracellular trafficking of NR1 via over-representation of the C2’ isoform.
DECREASED S-PALMITOYLATION OF PROTEINS AS A POTENTIAL MECHANISM FOR ABNORMAL RECEPTOR TRAFFICKING IN FRONTAL CORTEX IN SCHIZOPHRENIA James Meador-Woodruff, Janusz Tucholski, Anita Pinner Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL, USA Accumulating evidence suggests abnormalities of glutamate transmission in schizophrenia. While many findings of abnormal glutamate receptor expression in schizophrenia have been published, findings are subtle or even contradictory. These conflicting studies on the expression of these receptors leads to a reconsideration of the “glutamate hypothesis of schizophrenia” as not “too many” or “too few” receptors, but rather one of alterations in the cell biological processes that manage the total pool of receptors. We and others have recently published data pointing to abnormalities of glutamate receptor trafficking, delivery, dendritic localization, recycling, and degradation in the brain in schizophrenia. In this study, we begin to explore possible mechanisms to explain these abnormalities of intracellular trafficking of glutamate receptors in schizophrenia. Recent studies have found that synaptic proteins can be S-palmitoylated in a process of the thioesterification of the 16-carbon palmitoyl moiety to cysteine residues, and that palmitoylation regulates both trafficking and synaptic targeting of these proteins. We have adopted and validated a biotin switch assay to study S-palmitoylation of proteins in human postmortem brain tissue from subjects with schizophrenia and a comparison group. We identified a replicable pattern of palmityolated proteins in human brain that cluster into 17 bands of discrete molecular weight, which include based on mass spectrometry analysis numerous proteins associated with receptor signal transduction. The extent of S- palmitoylation was markedly reduced for the vast majority of proteins in most of the 17 molecular weight clusters that we assayed in schizophrenia. In rats treated for 9 months with haloperidol decanoate, the same pattern of palmitoylation was observed but the extent of palmitoylation was unchanged, suggesting that this profound diminution in extent of palmitoylation seen in schizophrenia is associated with the illness itself and not the effects of chronic treatment with antipsychotics. Conclusion: These results suggest that there are changes in the extent of S- palmitoylation of many proteins in the frontal cortex in schizophrenia. Given the roles of this posttranslational modification on synaptic trafficking and targeting of proteins associated with signal transduction, these data point to a potential mechanism explaining previous observations of abnormal trafficking of glutamate receptors in this illness. These data also suggest that there may be widespread abnormalities involving many other proteins in this illness, suggesting a hypothesis that we are currently pursuing of abnormal protein trafficking as part of the pathophysiology of schizophrenia.
Symposium INTERVENING IN PEOPLE AT-RISK OF PSYCHOSIS: WHAT IS THE EVIDENCE? Chairpersons: Patrick D. McGorry and Andreas Bechdolf Discussant: Jim van Os Sunday, 15 April 2012 4:15 pm – 6:15 pm Overall Abstract: Whether or not an attenuated psychotic symptoms syndrome should be included in DSM V is a widely and controversial discussed topic in the scientific community. Beside the predictive value of the proposed symptoms, the effects of indicated prevention efforts in this syndrome are the main issues in the discussion. In this symposium we present
Abstracts of the 3rd Biennial Schizophrenia International Research Conference / Schizophrenia Research 136, Supplement 1 (2012) S1–S375
to many cellular processes including the recycling of NMDA receptors and through this mechanism may contribute to the NMDA receptor hypofunction described in schizophrenia. It is responsible for the uptake of cargos as part of a clathin coated vesicles which, once endocytosed and uncoated form early endosomes. These can either recycle directly back to the plasma membrane, transfer to recycling endosomes, or enter the late endosomal/lysomal pathway for degradation. Apart from their role in CME, clathrin-interacting proteins are involved in a wide network of cellular processes, including endosomal sorting, lysosome biogenesis, antigen presentation, and cell migration. While trafficking changes in schizophrenia have been considered previously, a specific role for the clathrin interactome has not been considered. We integrate recent evidence implicating CME and related cellular trafficking mechanisms in the pathophysiology of psychotic disorders. Specifically, we review the proteomic and genomic literature, including our own data, for a potential role of CME, and membrane trafficking generally, in psychosis and we searched for evidence, linking CME with key features of psychosis pathophysiology, namely synaptic function, white matter integrity, and neurodevelopment, and studies linking the pharmacology of antipsychotic drugs with endocytic mechanisms. Proteomic and genomic findings implicate proteins and genes of the clathrin interactome. Additionally, several important candidate genes for schizophrenia, such as dysbindin, are involved in processes closely linked to CME and membrane trafficking. Furthermore, many antipsychotic drugs have been shown to affect clathrin-interacting proteins. Based on unbiased systems proteomics and genetic studies, we propose that there is now solid evidence for disturbances of proteins interacting with the membrane vesicle coat protein clathrin in psychosis (1). We propose that the targeted pharmacological manipulation of the clathrin interactome may offer fruitful opportunities for novel treatments of schizophrenia.
ALTERED INTRACELLULAR TRAFFICKING OF NR1 SUBUNIT IN SCHIZOPHRENIA Chang-Gyu Hahn, Anamika Banerjee, Matthew L. MacDonald, Karin E. Borgmann-Winter Department of Psychiatry and Pharmacology, University of Pennsylvania, Philadelphia, PA, USA Multiple lines of evidence support the premise of attenuated NMDA receptor (NR) function in schizophrenia (SCZ), yet the molecular underpinnings for such receptor dysfunction are largely unknown. Synaptic NR complexes reside in the postsynaptic density (PSD) and consist of NR subunits, scaffolding proteins and many other signaling proteins. Thus, the overall receptor function will be determined by the abundance of NR complexes in the PSD, their protein composition and post-receptor signaling activity. Despite multiple confounds, postmortem brains of patients may harbor disease specific characteristics. Evaluation of receptor activity in postmortem brains, however, has been a technical challenge. In this study, we evaluated NR complexes in the postmortem prefrontal cortex (PFC) of 14 pairs of SCZ subjects and their matched pairs with respect to the abundance and synaptic targeting of NR subunits. MK-801 is a non-competitive open channel blocker whose binding to NRs is relatively irreversible. Thus, MK-801 binding in synaptic membranes could reflect the abundance of membrane bound NR complexes. We found that Bmax for MK-801 was enhanced by about 30% (p≤0.017), while the dissociation constant, KD, was also increased in the SCZ group (p≤0.01). This increase in MK-801 binding could be accompanied by increases in NRs and NR complex proteins in the PSD. To address this, we obtained subcellular fractions enriched for the PSD (called PSD fractions henceforth). We found that NR1 subunit was significantly enhanced in the SCZ group compared to the control (p values ≤0.03), while NR2A and NR2B subunits and scaffolding proteins were unaltered. The increase in NR1 in the PSD could result from an up-regulation of the protein or from altered intracellular trafficking. We quantified NR1 levels in the whole tissue homogenate, synaptic membrane, vesicular and parasynaptic fractions. We found no between group differences of NR1 expression in all fractions except a trend for a decrease in para-synaptic fractions. Intracellular trafficking and subcellular localization of NR1 is tightly regulated by the expression of NR1 isoforms, C1, C2 and C2’. The C2’ isoform has higher affinity for PSD-95 and the NR1: PSD-95 association facilitates the insertion of the complex into the PSD. To test the role of C2’, PSD fractions derived from the PFC of patients and their matched controls
were immunoprecipitated for Pan’-NR1 and immunoblotted for NR1-C2’. We observed an increase in the C2’ splice variant of NR1 in schizophrenic samples with respect to pan NR1 (p≤0.04), suggesting that increased NR1 in the PSD and resultant increase in NR1: PSD-95 associations could be from an over-expression of C2’. Together, these results demonstrate that there are more NR complexes in synaptic membranes in SCZ, which is accompanied by altered intracellular trafficking of NR1 via over-representation of the C2’ isoform.
DECREASED S-PALMITOYLATION OF PROTEINS AS A POTENTIAL MECHANISM FOR ABNORMAL RECEPTOR TRAFFICKING IN FRONTAL CORTEX IN SCHIZOPHRENIA James Meador-Woodruff, Janusz Tucholski, Anita Pinner Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL, USA Accumulating evidence suggests abnormalities of glutamate transmission in schizophrenia. While many findings of abnormal glutamate receptor expression in schizophrenia have been published, findings are subtle or even contradictory. These conflicting studies on the expression of these receptors leads to a reconsideration of the “glutamate hypothesis of schizophrenia” as not “too many” or “too few” receptors, but rather one of alterations in the cell biological processes that manage the total pool of receptors. We and others have recently published data pointing to abnormalities of glutamate receptor trafficking, delivery, dendritic localization, recycling, and degradation in the brain in schizophrenia. In this study, we begin to explore possible mechanisms to explain these abnormalities of intracellular trafficking of glutamate receptors in schizophrenia. Recent studies have found that synaptic proteins can be S-palmitoylated in a process of the thioesterification of the 16-carbon palmitoyl moiety to cysteine residues, and that palmitoylation regulates both trafficking and synaptic targeting of these proteins. We have adopted and validated a biotin switch assay to study S-palmitoylation of proteins in human postmortem brain tissue from subjects with schizophrenia and a comparison group. We identified a replicable pattern of palmityolated proteins in human brain that cluster into 17 bands of discrete molecular weight, which include based on mass spectrometry analysis numerous proteins associated with receptor signal transduction. The extent of S- palmitoylation was markedly reduced for the vast majority of proteins in most of the 17 molecular weight clusters that we assayed in schizophrenia. In rats treated for 9 months with haloperidol decanoate, the same pattern of palmitoylation was observed but the extent of palmitoylation was unchanged, suggesting that this profound diminution in extent of palmitoylation seen in schizophrenia is associated with the illness itself and not the effects of chronic treatment with antipsychotics. Conclusion: These results suggest that there are changes in the extent of S- palmitoylation of many proteins in the frontal cortex in schizophrenia. Given the roles of this posttranslational modification on synaptic trafficking and targeting of proteins associated with signal transduction, these data point to a potential mechanism explaining previous observations of abnormal trafficking of glutamate receptors in this illness. These data also suggest that there may be widespread abnormalities involving many other proteins in this illness, suggesting a hypothesis that we are currently pursuing of abnormal protein trafficking as part of the pathophysiology of schizophrenia.
Symposium INTERVENING IN PEOPLE AT-RISK OF PSYCHOSIS: WHAT IS THE EVIDENCE? Chairpersons: Patrick D. McGorry and Andreas Bechdolf Discussant: Jim van Os Sunday, 15 April 2012 4:15 pm – 6:15 pm Overall Abstract: Whether or not an attenuated psychotic symptoms syndrome should be included in DSM V is a widely and controversial discussed topic in the scientific community. Beside the predictive value of the proposed symptoms, the effects of indicated prevention efforts in this syndrome are the main issues in the discussion. In this symposium we present